The present invention relates to a method for producing heterologous glycosylated proteins in bryophyte cells, such as in transformed Physcomitrella patens cells in culture. In particular, the method relates to a method for producing glycosylated proteins comprising animal glycosylation patterns, such as pharmaceutical proteins for use in mammals, including humans, in bryophyte cells such as Physcomitrella patens cells, the genetic material required therefore, such as DNA and RNA, vectors, host cells, methods of introduction of genetic material there into, and uses thereof.
In the past, heterologous proteins have been produced using a variety of transformed cell systems, such as those derived from bacteria, fungi, such as yeasts, insect, plant or mammalian cell lines (Kudo T. 1994, In: Y. Murooka and T. Amanaka (Eds.) Recombinant microbes for industrial and agricultural applications, pp. 291-299, Marcel Dekker, New York; Harashima S., Bioproc. Technol. 1994, 19: 137-158; Archer D. B. 1994, In: Y. Murooka and T. Amanaka (Eds.) Recombinant microbes for industrial and agricultural applications, pp. 373-393, Marcel Dekker, New York; Goosen M. F. A. 1993, In: M. F. A. Goosen, A. Baugulis and P. Faulkner (Eds.) Insect cell culture engineering, pp. 1-16, Marcel Dekker, New York; Hesse F. & Wagner R., Trends in Biotechnol. 2000, 18(4): 173-180).
Proteins produced in prokaryotic organisms may not be post-translationally modified in a similar manner to that of eukaryotic proteins produced in eukaryotic systems, e.g. they may not be glycosylated with appropriate sugars at particular amino acid residues, such as aspartic acid (N) residues (N-linked glycosylation). Furthermore, folding of bacterially-produced eukaryotic proteins may be inappropriate due to, for example, the inability of the bacterium to form cysteine disulfide bridges. Moreover, bacterially-produced recombinant proteins frequently aggregate and accumulate as insoluble inclusion bodies.
Eukaryotic cell systems are better suited for the production of glycosylated proteins found in various eukaryotic organisms, such as humans, since such cell systems may effect post-translational modifications, such as glycosylation of produced proteins. However, a problem encountered in eukaryotic cell systems which have been transformed with heterologous genes suitable for the production of protein sequences destined for use, for example, as pharmaceuticals, is that the glycosylation pattern on such proteins often acquires a native pattern, that is, of the eukaryotic cell system in which the protein has been produced: glycosylated proteins are produced that comprise non-animal glycosylation patterns and these in turn may be immunogenic and/or allergenic if applied in animals, including humans.
The use of recombinant glycoproteins produced by higher plants is limited by the plant-specific N-glycosylation that is acquired on such proteins. Compared to mammalian-derived glycoproteins, higher plant-specific glycoproteins contain two additional residues. Moreover, in higher plant glycoproteins terminal beta 1,4-galactose residues are not found, indicating that a beta 1,4-galactosyltransferase is not present in plants. Stable integration and expression of this enzyme in tobacco plants (Bakker et al. (2001) Proc Natl Acad Sci USA, 98, 2899-2904) as well as in tobacco BY2 cells (Palacpac et al. (1999) Proc Natl Acad Sci USA 96, 4692-4697) has been described. The recombinant human beta 1,4-galactosyltransferase was functional and proteins isolated from transgenic material exhibited terminal beta 1,4-galactose residues. Nevertheless, in higher plants it is not thought possible to suppress the activities of beta 1,2-xylosyltransferase and alpha 1,3-fucosyltransferase, the two enzymes that are considered responsible for transferring the additional, plant-specific residues. These residues are considered to be allergenic for humans (Garcia-Cassado et al. (1996) Glycobiology 6, 471-477; van Ree et al. 2000, J. Biol. Chem. 275, No. 15, 11451-11458). All data on plant-specific N-glycosylation has been generated in studies with higher plants.
The bryophyte, Physcomitrella patens, a haploid non-vascular land plant, can also be used for the production of recombinant proteins (WO 01/25456).
The life cycle of mosses is dominated by photoautotrophic gametophytic generation. The life cycle is completely different to that of the higher plants wherein the sporophyte is the dominant generation and there are notably many differences to be observed between higher plants and mosses.
The gametophyte of mosses is characterised by two distinct developmental stages. The protonema which develops via apical growth, grows into a filamentous network of only two cell types (chloronemal and caulonemal cells). The second stage, called the gametophore, differentiates by caulinary growth from a simple apical system. Both stages are photoautotrophically active. Cultivation of protonema without differentiation into the more complex gametophore has been shown for suspension cultures in flasks as well as for bioreactor cultures (WO 01/25456). Cultivation of fully differentiated and photoautrophically active multicellular tissue containing only a few cell types is not described for higher plants. The genetic stability of the moss cell system provides an important advantage over plant cell cultures and the stability of photoautotrophically active bryophyte cultures has been confirmed (Rieck 1996, Strukturaufklärung und stereochemische Untersuchungen von Sesquiterpenen als Inhaltsstoffe ätherischer Öle. Ph.D. thesis, Hamburg University). In cell cultures of higher plants the secondary metabolism is more differentiated and this results in differences in secondary metabolite profiles.
In addition, there are some important differences between mosses and higher plants on the biochemical level. Sulfate assimilation in Physcomitrella patens differs significantly from that in higher plants. The key enzyme of sulfate assimilation in higher plants is adenosine 5′-phosphosulfate reductase. In Physcomitrella patens an alternative pathway via phosphoadenosine 5′-phosphosulfate reductase co-exists (Koprivova et al. (2002) J. Biol. Chem. 277, 32195-32201). This pathway has not been characterised in higher plants.
Furthermore, many members of the moss, algae and fern families produce a wide range of polyunsaturated fatty acids (Dembitsky (1993) Prog. Lipid Res. 32, 281-356). For example, arachidonic acid and eicosapentaenoic acid are thought to be produced only by lower plants and not by higher plants. Some enzymes of the metabolism of polyunsaturated fatty acids, (delta 6-acyl-group desaturase) (Girke et al. (1998), Plant J, 15, 39-48) and a component of a delta 6 elongase (Zank et al. (2002) Plant J 31, 255-268), have been cloned from Physcomitrella patens. No corresponding genes have been found in higher plants. This fact appears to confirm that essential differences exist between higher plants and lower plants at the biochemical level.
Further differences are reflected in the regeneration of the cell wall. Protoplasts derived from higher plants regenerate new cell walls in a rapid manner, independently of the culture medium. Direct transfer of DNA via polyethylene glycol (PEG) into protoplasts of higher plants requires pre-incubation at 4 to 10° C. to slow down the process of cell wall regeneration (U.S. Pat. No. 5,508,184). In contrast, cell wall regeneration of protoplasts derived from protonema of Physcomitrella is dependent on culture medium. Protoplasts can be cultivated without regeneration of the cell wall over long periods. Without the intention of being bound by theory, it appears that the secretion machinery of the moss protoplast, essential for cell wall regeneration and protein glycosylation, differs from that of higher plants. Moreover, Physcomitrella patens shows highly efficient homologous recombination in its nuclear DNA, a unique feature for plants, which enables directed gene disruption (Girke et al. (1998) Plant J, 15, 39-48; Strepp et al. (1998) Proc Natl Acad Sci USA 95, 4368-4373; Koprivova (2002) J. Biol. Chem. 277, 32195-32201; reviewed by Reski (1999) Planta 208, 301-309; Schaefer and Zryd (2001) Plant Phys 127, 1430-1438; Schaefer (2002) Annu. Rev. Plant Biol. 53, 477-501) further illustrating fundamental differences to higher plants. However, the use of this mechanism for altering glycosylation patterns has proven to be problematic, as shown herein in the examples. Disruption of N-acetylglucosaminyltransferase I (GNT1) in Physcomitrella patens resulted in the loss of the specific transcript but only in minor differences of the N-glycosylation pattern. These results were in direct contrast to the loss of Golgi-modified complex glycans in a mutant Arabidopsis thaliana plant lacking GNT1 observed by von Schaewen et al. (1993) Plant Physiol 102, 1109-1118). Thus, the knockout in Physcomitrella patens did not result in the expected modification of the N-glycosylation pattern.
Although the knockout strategy was not successful for GNT1, the present inventors attempted to knock out the beta 1,2-xylosyltransferase (XylT) and alpha 1,3-fucosyltransferase (FucT) in Physcomitrella patens. Specific transcripts could not be detected in the resulting plants. Surprisingly, the N-linked glycans isolated from the transgenic plants were found to be modified in the desired manner. No 1,3 linked fucosyl residues could be detected on N-linked glycans of FucT knockout plants and no 1,2 linked xylosyl residues could be detected on N-linked glycans of XylT knockout plants. The isolated transgenic lines showed normal growth which is surprising considering that plant specific glycosylation is highly conserved and therefore would be expected to be significant for function. Double knockouts should therefore have been expected to have had a detrimental effect on the growth of the moss. In addition, compensating were expected but surprisingly were not apparent. Moreover, the double knockout of FucT and XylT resulted in modified N-linked glycans without detectable 1,3 linked fucosyl and 1,2 linked xylosyl residues.
Integration of the human beta 1,4-galactosyltransferase into the genome of a double knockout Physcomitrella patens plant resulted in a mammalian-like N-linked glycosylation pattern without the plant specific fucosyl and xylosyl residues and with mammalian-like terminal 1,4 galactosyl residues. The galactosyltransferase was found to be active. Such a modification of N-linked glycans without loss of viability was not expected because N-glycosylation is very complex and well regulated. It is not only dependent on developmental stages (for plants: Elbers et al. (2001) Plant Phys 126, 1314-1322) but also dependent on culture conditions (for mammalian cell culture: Hills et al. (2001) Biotechnol. Bioeng. 75, 239-251).
It is an object of the present invention to provide a more efficient method of producing animal proteins comprising animal glycosylation patterns, and in particular, glycosylated human proteins comprising human glycosylation patterns thereon. It is a further object to provide an efficient process for the production of heterologous animal proteins comprising animal glycosylation patterns, particularly human proteins comprising human glycosylation patterns in bryophytes, such as Physcomitrella patens. 
These and other objects will become apparent from the following description and examples provided herein.